This invention relates to high-throughput screening using small-animal models. This technology can be applied to study neurotoxicity, neural degeneration, regeneration, neurological disorders, Alzheimer's disease, Parkinson's disease, wound healing, the immune system, metabolism, aging, development, stem cells, reproduction, heart diseases, vascular, liver, kidney, bladder, intestinal or tooth development. Potential applications include drug screening and discovery, population enrichment, genetic screening, and target and lead validation.
Existing large vertebrate animal models currently cannot be used in high-throughput assays for rapid identification of new genes and drug targets because of the size and complexity of the instrumentation with which these models are studied. In recent years, the advantages of using small invertebrate animals as model systems for human disease have become increasingly apparent, and have resulted in two Nobel Prizes in Physiology or Medicine during the last six years for studies conducted on the nematode C. elegans. The availability of a wide array of species-specific genetic techniques, along with the worm's transparency, and its ability to grow in minute volumes make C. elegans an extremely powerful model organism. The use of small vertebrate animals is exemplified in studies of zebrafish (D. rerio). Like C. elegans, zebrafish are transparent, develop quickly, and a wide range of genetic controls are available. Additionally, being vertebrates, zebrafish are more closely related genetically to humans, thus increasing the likelihood that any discovered process is conserved between the two.
However, the techniques to manipulate both C. elegans and zebrafish have not evolved with their respective fields. These manipulations are primarily manual, and performed on individual worms or fish. As a result, large-scale assays such as mutagenesis and reverse genetic screens (1-3) can take months or even years to complete manually. For example, high-throughput C. elegans assays are currently performed by adapting techniques developed for screening cell lines, such as flow-through sorters and microplate readers (4-6). Due to the significant limitations of these methods, high-throughput small-animal studies either have to be dramatically simplified before they can be automated or cannot be conducted at all. The numbers in parentheses refer to the references appended hereto. The contents of all of these references are incorporated herein by reference.
Existing small-animal sorters such as the BIOSORT and XL from COPAS use a flow-through technique similar to the fluorescence-activated cell sorter (FACS) technology. These systems can capture and analyze only one-dimensional intensity profiles of the animals being sorted and as a result, three-dimensional cellular and sub-cellular features cannot be resolved (13).
Although microfluidics have previously been used to perform novel assays on C. elegans, so far research has been limited to specific applications such as generation of oxygen gradients (7), worm culturing/monitoring during space flight (8), optofluidic imaging (9) and maze exploration (10). See also, U.S. Pat. No. 6,400,453.
Heretofore, accurate control of the nematode's or fish's microenvironment has not been possible. Such microenvironment control is necessary for making quantitative, artifact free, and repeatable measurements. Conventional techniques involve many manual steps wherein worms are exposed to different temperatures, abrupt and inaccurately timed changes. Manual handling is also error prone. All of these factors not only slow down assays dramatically, but also cause artifact errors.
It is therefore an object of the present invention to provide a high-throughput screening system for use with small-animal models.